The invention relates to the field of biological microscopy, in particular to two-color-fluorescence localized super-resolution biological microscopy method and system.
Since Ernst Abbe proposed the theory of optical imaging resolution limit in the 1770s, people have been looking for ways to break the resolution limit. Through single molecule fluorescence localization, Zhuang Xiaowei and Eric Betzig successively put forward Stochastic Optical Reconstruction Microscopy (STORM) and Photoactivated Localization Microscopy (PALM) respectively in 2006 realizing super-resolution of ten times the optical resolution limit. Eric Betzig won the Nobel Prize in Chemistry 2014 by the PALM. The fluorescence localization microscopies have been partially commercialized, and applied to researches of fundamental life science, especially molecular biology and biochemistry. Researchers can investigate detailed structures of biological samples at lateral resolution of 10 to 20 nm and vertical resolution of 50 nm by these techniques. Compared with other existing super-resolution techniques such as electron microscopy, the fluorescence localization microscopies greatly simplify the preparation process of samples, and can realize the live-cell imaging. What's more, the fluorescence localization microscopies can easily realize multi-channel co-localization imaging, and thus obtain the interaction between proteins, providing the most immediate evidences for a large number of molecular bioresearch topics. However, the performance of existing techniques in multi-color super-resolution imaging still has shortcomings. As a representation of the commercial fluorescence localization microscopies, two-channel imaging of Nikon N-STORM uses fluorescent switches combining two fluorescent molecules (e.g., Alexa647-Alexa488 molecular pair and Alexa467-Alexa405 molecular pair) with different excitation wavelengths as markers. The method can realize quick switch between different imaging channels, but the markers of such fluorescent switches are not currently commercialized, and preparation thereof in laboratory is relatively complicated. What's more, the two-color imaging method will produce severe channel crosstalk, and produce wrong co-localization information. Leica SR GSD uses ordinary fluorescent molecules (e.g., Alexa647 and Alexa532) with different excitation wavelengths as markers, and uses a filter system to realize two-channel imaging. The limitation of the method is that fluorescent molecules with short excitation wavelengths are easy to be photo-bleached, and the short excitation wavelengths will lead to autofluorescence background of cells, which affects the imaging quality.
In addition, sample drift is a common problem in super-resolution fluorescence localization microscopies, which means that samples will move tens to hundreds of nanometers during shooting due to environmental instability such as airflow, temperature change and noise. Although the drift is common in microscopy systems, the drift is not obvious as the resolution of an ordinary microscope is less than 300 nm. But for a super-resolution microscope with resolution up to dozens of nanometers, the drift will severely interfere with imaging. Most of existing solutions are to add fluorescent particles in samples, record displacement of the particles in imaging, and subsequently subtract the displacement from obtained super-resolution images. The defects are difficult preparation and the fluorescent particles will also occupy an imaging channel. Besides, fluorescence of the fluorescent particles will attenuate over imaging time due to photo-bleaching, therefore, the correction precision of drift gets worse over time.
To sum up, existing techniques have obvious inconvenience and defects in practice, so improvement is necessary.